Fluorescence is the phenomenon in which light of a given wavelength is absorbed by a fluorescent molecule (e.g., a “fluorophore”), thereby resulting in emission of light at longer wavelengths. The distribution of the wavelength-dependent intensity that causes fluorescence is known as the fluorescence excitation spectrum, and the distribution of wavelength-dependent intensity of emitted energy is known as the fluorescence emission spectrum.
Using fluorescence, one can monitor minute changes in the concentration of a substance. Changes in fluorescence intensity on the order of picoseconds can be detected if necessary. Over the past decade, investigators have proposed many new applications for fluorescence spectroscopy in the physical and life sciences in view of advances in time resolution, methods of data analysis, and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of macromolecules, to probe the structure of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry.
In a fluorescence spectrometer, the sample to be analyzed is irradiated by excitation light, which causes the sample to emit fluorescence light at characteristic wavelengths. The fluorescence light is measured by a suitable detector to derive information about the sample, in particular the composition of the sample and the quantities of the individual components present in the sample. Typically, the wavelength of the excitation light is adjusted by an optical component, such as a diffraction grating or a filter. The fluorescence light emitted is usually analyzed by a second diffraction grating or by a filter. For performing a fluorescence measurement, the grating at the excitation side of the spectrometer is set to a fixed excitation wavelength and the wavelength spectrum of the fluorescence light is recorded by means of the grating at the emission side (emission grating). The emission spectrum can be recorded for a plurality of excitation wavelengths. As an alternative thereto, the emission wavelength can be kept fixed and the excitation wavelength can be varied by corresponding adjustment of the excitation grating.
Laser induced fluorescence spectroscopy has heretofore been used to determine the chemical composition of, or pathological conditions in, biological tissue. For example, U.S. Pat. Nos. 5,419,323 (Kittrell et al.) and 5,562,100 (Kittrell et al.) describe methods for laser induced fluorescence of tissue in which laser radiation is used to illuminate and induce fluorescence in the tissue for the purpose of determining the chemical composition of, or a pathologic condition in, the tissue. The laser radiation and the retrieved fluorescing radiation can be conveyed through a catheter using an array of optical fiber. The fluorescence spectrum of the tissue can be displayed and analyzed to obtain information regarding the chemical composition and medical condition of the tissue inside the human body. Also, U.S. Pat. No. 5,337,676 (Vari et al.) describes a method for determining the biodistribution of substances using fluorescence spectroscopy wherein a photosensitizing agent or other intrinsically fluorescent agent, or an agent labeled with an extrinsic fluorophore, is administered to a subject. A fiberoptic probe integrated with an excitation light source illuminates the tissue and causes fluorescence. The fluorescence is recorded by a spectrograph and plotted as a spectral curve. The intensity ratio (S1/S2) for the fluorescence from the photosensitizing agent (S1) and autofluorescence (S2) for the examined tissue is used as an index for drug presence and compared with the intensity ratio at the same wavelengths for various tissues.
Laser induced fluorescence spectroscopy has also been used to determine the presence of certain chemicals or substances within non-biological materials. For example, U.S. Pat. No. 5,198,871 (Hill Jr., et al.) describes an optical inspection system wherein laser-induced luminescence is used to determine the quality of materials, such as fuel. The inspection system comprises an excitation means, such as a laser, for illuminating a specimen and for causing the specimen to produce fluorescent radiation. The fluorescence spectrum produced by the specimen is then compared to a reference spectrum to obtain an indication of the physical characteristics of the specimen (e.g., to determine the presence of chemical impurities or degradation products within the specimen).